Characterization of N-linked oligosaccharides bearing sialyl Lewis x moieties on an alternatively glycosylated form of soluble complement receptor type I (sCRI)

We sought to produce a complement inhibitory protein possessing oligosaccha rides specifically modified to contain the sialyl Lewis x (sLe(x)) moiety. This modified glycoprotein could combine anti-complement activity with the ability to inhibit selectin-mediated interactions and concentrate this acti vity to sites of activated endothelium where selectins are upregulated, Sol uble complement receptor type I (sCRI), previously shown to be effective in inhibiting the complement cascade, was produced in a cell line capable of adding fucose to N-linked oligosaccharides in the alpha I-3 linkage, which is necessary for sLe(x) glycosylation. The glycoprotein purified from these cells was designated sCRIsLe(x), and may prove to be more effective than s CRI in some clinical applications. Detailed analysis and characterization o f sCRIsLe(x) was performed to confirm that the N-linked oligosaccharides po ssessed sLe(x) moieties and also to determine the extent of sLe(x) glycosyl ation. The glycoproteins were characterized by oligosaccharide profiling, s equencing, linkage analysis and quantified by differential enzymic digestio n, using fluorophore-assisted carbohydrate electrophoresis. The major glyca ns were identified as biantennary oligosaccharides (including sialylated an d non-core fucosylated glycans), The linkages of sialic acid and the branch ed fucose were analysed by digestion with linkage-specific enzymes and subs equent separation by electrophoresis. All data were consistent with the pre sence of sLe(x) moieties on the N-linked oligosaccharides of sCRIsLe(x). sC RIsLe(x) is a prime example of a recombinant protein expressed with oligo-s accharides engineered for a specific biological function, and produced usin g a commercially viable method.